Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The invention relates to yeasts expressing the gene BDH1 coding for Bdh1p,
characterised in that they catalyse, in an alcoholic fermentation medium,
the reduction of acetoin into 2,3-butanediol by a rate at least twice
higher than that of the initial stem. The invention can particularly be
used for the fermentation of fruit juice and for producing
2,3-butanediol.

Claims:

1. A yeast strain in which the BDH1 gene encoding Bdh1p is overexpressed
relative to the initial strain, characterized in that it catalyzes the
reduction of acetoin to 2,3-butanediol according to a rate that is at
least twice that of the initial strain.

2. A yeast strain comprising the BDH1 gene encoding Bdh1p, characterized
in that it is a strain genetically transformed so as to overexpress BDH1,
by means of a strong yeast promoter.

3. The strain as claimed in claim 1, characterized in that the BDH1 gene
comprises one or more mutations in such a way as to encode a Bdh1p
protein in which one or more amino acids are mutated.

4. The yeast strain as claimed in claim 1, characterized in that it
comprises one or more mutations in the BDH1 gene in order to exhibit a
cofactor specificity for NADPH instead of NADH.

5. The yeast strain as claimed in claim 3, characterized in that the
protein encoded by the mutated BDH1 gene comprises 3 mutations at
position 221, 222 and 223, corresponding to a 221 SRS 223 fragment with
reference to the Saccharomyces cerevisiae S288C strain.

6. The yeast strain as claimed in claim 1, characterized in that it is
genetically transformed and also comprises one or more mutations in the
BDH1 gene in order to exhibit a cofactor specificity for NADPH instead of
NADH.

7. The yeast strain as claimed in claim 1, characterized in that it
overproduces glycerol.

8. The yeast strain as claimed in claim 7, characterized in that it
comprises an overexpressed GPD1 gene or an overexpressed GPD2 gene.

9. The yeast strain as claimed in claim 1, characterized in that it is a
genetically modified strain capable of reducing the amount of acetate
produced in comparison with the strain as claimed in claim 8.

10. The yeast strain as claimed in claim 9, characterized in that the ALD6
gene has been deleted, as have, where appropriate, copies thereof.

11. The yeast strain as claimed in claim 1, characterized in that it
belongs to the Saccharomyces genus.

12. The yeast strain as claimed in claim 11, characterized in that it is
Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces uvarum or
Saccharomyces kudriavzevii.

13. The yeast strain as claimed in claim 1, characterized in that it
belongs to the non-Saccharomyces genus.

14. The yeast strain as claimed in claim 11, characterized in that it is a
hybrid.

15. A method for reducing the amount of acetoin in a yeast strain,
characterized in that it comprises the expression of a yeast strain as
claimed in claim 1.

16. A method for fermenting fruit juice, characterized in that it
comprises the addition, to a fermentation medium, of yeast strains as
defined in claim 1.

17. The method as claimed in claim 16, characterized in that the fruit
juice is grape juice.

18. The use of the yeast strains as claimed in claim 1, for producing
2,3-butanediol.

19. The use of the strains as claimed in claim 1, for reducing diacetyl
production.

Description:

[0001]The invention relates to yeasts and to fermentation methods using
these yeasts, for reducing acetoin buildup in alcoholic fermentation
media.

[0002]Over the past fifteen years or so, scientific knowledge and know-how
in viticulture and enology have led to a very significant improvement in
the organoleptic qualities of wines. Current wine-making practices favor
the production of wines with a high qualitative potential by delaying the
time of the grape harvest. A major consequence of this is an increase in
the sugar content of the musts, and therefore in the alcohol content of
the wines (frequently greater than 14°. This drift, encountered in
most producing regions, openly poses many problems for the wine-making
industry throughout the world. Excessive alcohol contents are in fact not
very compatible with the preoccupations of health and of well-being of
consumers, and are, moreover, the subject of taxation in certain
countries.

[0003]As a result, there is an increasing demand for methods and tools
that make it possible to reduce the alcohol content of wines and other
alcoholic beverages. Physical approaches (for example, vacuum
distillation) are increasingly used, but are not very compatible with the
maintaining of satisfactory organoleptic quality.

[0004]One biological solution would be based on the use of yeast strains
with a low alcohol yield.

[0005]For example, Saccharomyces cerevisiae yeasts, in particular
enological Saccharomyces cerevisiae yeasts, convert sugars to alcohol
with a yield of 0.47 g/g, which varies little according to the strain
used. As a result, the production of a Saccharomyces cerevisiae yeast
with a low alcohol yield requires the implementation of genetic
strategies aimed at diverting a part of the sugars to the formation of
other by-products.

[0006]Several genetic engineering approaches have been implemented in
order to divert a part of the sugars to the production of by-products
other than ethanol. These approaches have been based on the modification
of the activity of enzymes involved in the synthesis of glycerol or in
the use of pyruvate. For example, the overproduction of glycerol,
obtained by overexpression of GPD1 or GPD2 encoding glycerol-3-phosphate
dehydrogenase (Michnick et al., 1997; Remize et al., 1999; international
patent application WO 96/41888) has thus made it possible to reduce the
ethanol content by to 2°. It is, however, accompanied by major
modifications of the level of production of other metabolites, including
some which are undesirable in wine, especially acetate and acetoin
(3-hydroxy-2-butanone). The production of acetate can be reduced by
deleting ALD6 which encodes an acetaldehyde dehydrogenase. On the other
hand, no strategy has for the moment been described for reducing acetoin
buildup. This compound builds up at a rate of several grams per liter,
although it is unfavorable, for example in wine, at more than 150 mg/l
(olfactory detection threshold).

[0007]The solution proposed by the inventors consists in converting the
acetoin that is produced into 2,3-butanediol, a compound considered to be
neutral from an organoleptic point of view, the detection threshold
thereof in wine being greater than 12 g/l.

[0008]The production of 2,3-butanediol from acetoin is carried out by the
butanediol dehydrogenase (Bdh1p) enzyme encoded by BDH1.

[0009]The 2,3-butanediol production pathway contributes to the
intracellular NAD.sup.+/NADH redox balance. In bacteria, this pathway can
also be involved in the regulation of intracellular pH.

[0010]2,3-Butanediol is produced in several forms: (2R,3R)-2,3-butanediol
and (2S,3S)-2,3-butanediol, two optically active forms,
(2R,3S)-2,3-butanediol and (2S,3R)-2,3-butanediol, corresponding to the
meso forms.

[0011]The butanediol dehydrogenase encoded by BDH1 is the main enzyme
involved in the production of 2,3-butanediol. It is responsible for the
formation of all the (2R,3R)-2,3-butanediol and a part of the
(meso)-2,3-butanediol, from R-acetoin and S-acetoin, respectively. Since
Bdh1p has, moreover, a greater affinity for acetoin (Kmacetoin: 4.5
mM, Km2,3-butanediol: 14 mM), the reaction is strongly shifted in
the direction of the formation of 2,3-butanediol.

[0012]In the approach followed, the inventors have considered that the
level of synthesis of 2,3-butanediol dehydrogenase is a factor that
limits the reaction for conversion of acetoin to 2,3-butanediol. They
have also retained the fact that a reduced availability of NADH could
prevent a more effective conversion of acetoin. A strategy of
overexpression of BDH1 and of site-directed mutagenesis of BDH1 has
therefore been implemented in order to change its cofactor specificity
from NADH to NADPH.

[0013]Their studies have thus made it possible to transform Saccharomyces
strains and to render them capable of efficiently converting acetoin to
2,3-butanediol over the course of an alcoholic fermentation, even when
the acetoin is produced in large amounts (several g/l).

[0014]The objective of the invention is therefore to provide novel yeast
strains genetically transformed so as to overexpress BDH1 and exhibiting
a modified, cofactor specificity, in order to produce larger amounts of
2,3-butanediol from acetoin.

[0015]The invention is also directed toward providing a method for
obtaining such strains.

[0016]The objective of the invention is also to take advantage of the
properties of these transformed yeast strains in a method of alcoholic
fermentation and also for the production of 2,3-butanediol.

[0017]The yeast strains of the invention are yeasts which overexpress,
relative to the initial strain, the BDH1 gene encoding Bdh1p.

[0018]These strains are characterized in that they catalyze, in an
alcoholic fermentation medium, the reduction of acetoin to 2,3-butanediol
according to a rate that is at least twice that of the initial strain.

[0019]They are more especially strains that have been genetically
transformed and/or mutated in such a way as to obtain an
acetoin-to-2,3-butanediol conversion rate that is at least twice that of
the initial strain.

[0020]The term "initial strain" is intended to mean a strain before
overexpression or modification of the BDH1 gene.

[0021]The invention is thus directed toward yeast strains as defined
above, genetically transformed so as to overexpress BDH1, by means of
regulatory sequences suitable for increasing the expression of said gene.
Such transformed strains contain at least 2 copies of the BDH1 gene. Any
promoter that is active in the host in which it is desired to obtain the
expression of this gene may be used, preferably promoters described as
strong (encoding strongly expressed genes) under alcoholic fermentation
conditions. This is the case, for example, of glycolytic genes strongly
expressed in fermentation, such as ADH1 (alcohol dehydrogenase), PGK1
(phosphoglycerate kinase) or TDH3 (glyceraldehyde dehydrogenase), or the
TEF1 transcription factor gene, but many others also exist.

[0022]The invention is also directed toward strains in which the BDH1 gene
comprises one or more mutations in such a way as to encode a Bdh1p
protein in which one or more amino acids are mutated.

[0023]Advantageously, such strains comprise one or more mutations in the
BDH1 gene in order to exhibit an increased affinity for the NADPH
cofactor instead of NADH.

[0024]The choice of amino acids to be modified is based on the fact that
NAD(H) differs from NADP(H) in terms of the phosphate group esterified at
the 2'-position of the ribose of adenosine. As a result, the amino acids
that interact with this group are candidates for the cofactor specificity
change. The residue which determines the NAD(H) specificity is aspartate
(Asp) or glutamate (Glu), which form hydrogen bonds with the 2'- and
3'-hydroxyl groups in the ribosyl part of the coenzyme. NADP(H)-dependent
dehydrogenases have a smaller and neutral residue, such as glycine (Gly),
alanine (Ala) and serine (Ser), at the same position. In addition, an
adjacent arginine residue (Arg) enables good interaction with the
phosphate group of NADP(H).

[0025]Thus, advantageous replacements in accordance with the invention
concern the Glu(E)221 residue replaced with Ser(S), the Ile(I)222 residue
replaced with Arg(R) and the Ala(A)223 residue replaced with Ser, using
as a basis the structure of the NADPH-dependent Adh6p enzyme of S.
cerevisiae (the positions are denoted according to the amino acid
sequence of Bdh1p of Saccharomyces cerevisiae S288C).

[0026]The modification(s) of Glu(Asp)221 and/or Ile(val)222 and/or Ala223
and any possible combination of these three modifications are also part
of the field of the invention.

[0027]The first residue may also be a serine (as in the example), or any
small, neutral amino acid such as Gly or Ala.

[0028]The invention is also directed toward strains as defined above,
genetically modified so as to overexpress BDH1 in an alcoholic
fermentation medium, by means of a strong yeast promoter, and comprising
one or more mutations in BDH1 in order to exhibit a cofactor specificity
for NADPH instead of NADH.

[0029]In one preferred embodiment of the invention, the yeast strains
which produce acetoin are strains that overproduce glycerol. They are in
particular strains containing the overexpressed GPD1 gene or the
overexpressed GPD2 gene, for example obtained by transformation of the
strains with the pVT100U-ZEO-URA3-GPD1 plasmid or by in situ exchange of
its promoter with a strong promoter.

[0030]In another embodiment of the invention, the yeast strains are
genetically modified in such a way as to reduce the production of acetate
(in addition to the overexpression of GPD1). More particularly, they are
strains in which the ALD6 gene and, where appropriate, copies thereof has
(have) been deleted.

[0031]Preferred strains belong to the Saccharomyces genus and comprise, in
particular, the species Saccharomyces cerevisiae, Saccharomyces bayanus,
Saccharomyces uvarum and Saccharomyces kudriavzevii.

[0032]Other preferred strains belong to the non-Saccharomyces genus.

[0033]The invention is also directed toward the hybrids of the strains
defined above.

[0034]The invention is also directed toward a method for obtaining the
yeast strains mentioned above.

[0035]This method advantageously takes advantage of the genetic
engineering and site-directed mutagenesis techniques. By way of example,
mention will be made of the use of an oligonucleotide comprising the
desired target mutations in order to modify the BDH1 gene of a yeast
strain, and the transformation of the strain with an amplified fragment,
the amplification of a region of the gene comprising the mutations, or
alternatively crossing between strains, starting from a strain
comprising, for example, the desired mutations in order to transfer them
into another.

[0036]A subject of the invention is also a method of fermentation,
characterized by the addition of a transformed yeast strain as defined
above to the fermentation medium.

[0037]The fermentation medium is in particular a must, advantageously a
grape juice.

[0038]According to one aspect of great importance, the 2,3-butanediol
obtained using the yeast strains of the invention constitutes an
important compound for a variety of chemically based materials and liquid
fuels.

[0039]This compound may result, by dehydration, in the formation of methyl
ethyl ketone, that can be used as a liquid fuel additive.

[0040]The 2,3-butanediol may also be converted to 1,3-butanediene, a
compound that is used in the production of synthetic gum. Other
derivatives, for uses, for example, as antifreezes (levo form), solvents
and plastics, may also be prepared from 2,3-butanediol. In addition, it
may be added to food products as a flavor after conversion to diacetyl by
dehydrogenation.

[0041]The esterification of butanediol leads to the formation of
polyurethane precursors for use in medicaments, cosmetic products and
lotions, etc.

[0042]The use of the yeast strains for producing 2,3-butanediol, in
particular in the applications mentioned above, is also part of the field
of the invention.

[0043]In particular, the invention provides means for obtaining a higher
2,3-butanediol yield and productivity in general, but also for obtaining
pure stereoisomers, in large amounts, instead of a mixture of isomers.

[0044]The yeast strains of the invention also make it possible to reduce
the amount of diacetyl produced. They are advantageously used to this
effect, in particular in fermented beverages.

[0045]Other characteristics and advantages of the invention are given in
the exemplary embodiments which follow. In these examples, reference is
made to FIGS. 1 to 7 which represent, respectively:

[0046]FIG. 1: the effect of the overexpression of BDH1 on the production
of acetoin by the V5ald6 BDH1 pGPD1 strain for varying glycerol
concentrations;

[0047]FIG. 2: the production of acetoin (a and a') and of 2,3-butanediol
(b and b') over the course of fermentation for the V5 and V5ald6 pGPD1
and V5ald6 BDH1 pGPD1 strains under conditions (i): a, b and (ii): a',
b';

[0048]FIG. 3: sequence alignment of BDH1 (in bold) and BDH1223. In
gray: the change of 3 amino acid residues E221S/V222R/A223S inducing a
reversion of the cofactor specificity in Bdh1p;

[0049]FIG. 4: acetoin formation in the V5ald6 BDH1 pGPD1 (black) and
V5ald6 BDH1223 pGPD1 (white) strains over the course of a
fermentation;

[0050]FIG. 5: the final formation of acetoin in the CEN.PKald6 pGPD1
(white), CEN.PKald6 BDH1 pGPD1 (gray) and CEN.PKald6 BDH1223 pGPD1
(black) strains at the end of several fermentations;

[0051]FIG. 6: the functioning of mutant forms in vivo; and

[0052]FIG. 7: the reduction of diacetyl formation by overexpression of
BDH1.

[0054]A fragment constituted of the kanMX module, which carries the kanR
gene conferring resistance to the G418R antibiotic, and of the TDH3
promoter was amplified by PCR from the bacterial plasmid pUG6-NOXE (Heux
et al., 2005) using the oligonucleotides having the sequences,
respectively, SEQ ID No. 1 and SEQ ID No. 2, carrying a sequence
homologous to pUG6-NOXE and a flanking sequence (in italics) homologous
to the chromosomal target region (BDH1 promoter).

[0056]The PCR product obtained was precipitated with ethanol in the
presence of salts. 3 μg of the precipitated DNA were used to transform
the S. cerevisiae yeast strains V5 and V5ald6. The V5 strain (MATa, ura3)
is derived from an enological strain. The V5 strain and the V5ald6 strain
(Remize et al., 1999) in which the two copies of the ALD6 gene were
deleted were transformed by the lithium acetate method (Schiestl and
Gietz, 1989). The transformants were selected on YPD G418R dishes.
The integration of the TDH3 promoter in place of the BDH1 promoter was
verified by PCR.

Results

[0057]The results obtained show that the V5 BDH1 strain has the same
growth and fermentation rate as the V5 control strain. The measurement of
the BDH activity during the exponential phase and the stationary phase
shows that the BDH enzymatic activity of the strain overexpressing BDH1
is approximately 30 times greater than that of the wild-type strain
(average specific activity V5:0.1 U/mg total protein; average specific
activity V5 BDH1:3.2 U/mg total protein).

[0058]Effect of the Overexpression of BDH1 in Strains Overexpressing GPD1

[0062]The fermentations were carried out in 1.2-liter reactors (SGI,
France) with a reaction volume of 1 liter. The MS medium was used for
preculturing and culturing. It is a synthetic medium which simulates a
standard grape must (Bely et al., 1990). The MS medium contains 20%
glucose, 6 g/l of malic acid, 6 g/l of citric acid, and 460 mg/l of
nitrogen, in the form of NH4Cl (120 mg/l) and of amino acids (340
mg/l). The medium is supplemented with methionine (115 mg/l) and, if
necessary, uracil (50 mg/l). The pH of the MS medium is 3.3. Anaerobiosis
factors, ergosterol (7.5 mg/l), oleic acid (2.5 mg/l) and Tween 80 (0.21
g/l) are added. The precultures were prepared in 100 ml Erlenmeyer flasks
containing 20 ml of medium at 28° C. with shaking (150 rpm) for 30
h. The reactors were inoculated using these precultures, at a cell
density of 1×106 cells/ml, and maintained at a constant
temperature of 28° C. with continuous shaking (300 rpm).

[0063]The culture samples were collected using a syringe. The fermentation
data are expressed as a function of time.

[0064]Analytical methods--The growth was monitored by measuring the
optical density at 600 nm and by counting the number of cells on a
Coulter Counter instrument (ZBI) using an aliquot fraction of the culture
medium.

[0065]The metabolites were assayed in the supernatant, after
centrifugation, of the samples taken, at 13 000 rpm for 5 minutes. The
glucose, glycerol, ethanol, pyruvate, succinate, acetate,
α-ketoglutarate and 2-hydroxy-glutarate concentrations were
determined by high pressure liquid chromatography (HPLC) using an HPX-87H
column (Bio-Rad). The acetaldehyde concentration was determined by the
enzymatic method described by Lundquist (1974). The acetoin and
2,3-butanediol concentrations were determined by gas chromatography as
described by Michnick et al., 1997.

[0066]FIG. 1 shows the impact of the overexpression of BDH1 on acetoin
production. In this example, the production of this compound was analyzed
over the course of fermentation with V5ald6 pGPD1 and V5ald6 BDH1 pGPD1.
Two different conditions of glycerol overproduction were used. Under
conditions (i), no selection pressure was used to maintain the
pVT100U-ZEO-GPD1 plasmid, resulting in a moderate overproduction of
glycerol, reaching 10-15 g/l. Under conditions (ii), the V5 BDH1 pGPD1
and V5ald6 BDH1 pGPD1 strains were cultured in the absence of uracil,
enabling the plasmid to be maintained throughout the fermentation,
generating a very high overproduction of glycerol, 20-30 g/l.

[0067]Under the two conditions tested, (i) and (ii), the overexpression of
BDH1 allows a drastic decrease in acetoin production of up to 90%
relative to the level produced by the V5ald6 pGPD1 control strain, and a
corresponding increase in the production of 2,3-butanediol (FIG. 2). The
V5 strain was used as a control. This strain does not build up acetoin
and produces approximately 0.5 g/l of 2,3-butanediol.

[0068]FIG. 2 shows the acetoin levels obtained after several fermentation
experiments with the V5ald6 pGPD1 and V5ald6 BDH1 pGPD1 strains, under
the same conditions as above.

In Vitro Site-Directed Mutagenesis of BDH1

[0069]Starting from the protein sequence encoded by the BDH1 gene of the
S288C strain [Saccharomyces genome database http://www.yeastgenome.org/],
mutations corresponding to the change of one amino acid were introduced
at each PCR step. By means of oligonucleotides comprising the desired
mutations and a plasmid containing the native BDH1 of S288C (pYES2-BDH1),
the following mutated enzymes were constructed:

[0071]The mutations were introduced using a method based on the
Quickchange II XL Site-Directed Mutagenesis Kit

[0072](Stratagene, USA). The E221S mutant was constructed from the coding
region of BDH1 of the laboratory strain S288C cloned into the pYES2
plasmid (Invitrogen, Carlsbad, Calif.): pYES2-BDH1 (E. Gonzalez, 2000).
This shuttle vector contains an inducible promoter (promoter and UAS
sequence of GAL1), the 2μ origin of replication, the URA3 selectable
marker and the bacterial elements (origin of replication and
ampicillin-resistance gene).

[0073]The pYES2-BDH1 plasmid containing the E221S, E221S/I222R and
E221S/I222R/A223S mutations was used as a template to obtain the single
mutant, the double mutant and the triple mutant, respectively.

[0075]Escherichia coli cells (ultracompetent XL-10 Gold®, Stratagene,
La Jolla, Calif.) were transformed successively with the constructed
plasmids. The transformants were cultured at 37° C. in 5 ml of LB
medium supplemented with 50 μg/ml of ampicillin, and the plasmid DNA
of these clones was extracted with the Genelute Plasmid Miniprep® kit
(Sigma, USA) and analyzed by sequencing.

[0078]The absence of uracil makes it possible to maintain a selection
pressure for the plasmids.

[0079]Culture Conditions

[0080]20 ml of YNB medium (20% galactose) were inoculated with 5
transformants, respectively. The culturing was carried out at 28°
C. for 2 days.

[0081]Enzymatic Activity Assay

[0082]The butanediol dehydrogenase activity was determined using the crude
protein extracts at 25° C., by measuring the change in absorbance
at 340 nm. One activity unit corresponds to 1 μmol of cofactor used
per min, based on an absorption coefficient of 6220 cm-1 M-1 at
340 nm for NADH and NADPH. The optical paths for the reduction reactions
were 1 cm, 0.5 cm and 0.2 cm. The enzymatic activity assays were carried
out in the presence of 50 mM of acetoin in 33 mM NaH2PO4, pH
7.0/NaOH. The kinetic parameters were obtained by means of assays of
activity with coenzyme concentrations of 1/3×Km to
10×Km of BDH for NADH (0.055 mM).

[0083]The BDH activity was determined on crude extracts of the strain
expressing the wild-type form (pYES2-BDH1) and of the strains expressing
the mutated forms. The kinetic parameters of the various forms were also
determined (table 2).

[0084]The three mutants exhibit an affinity for NADH that is very greatly
reduced, by 90% on average relative to that of the native enzyme.

[0085]A substantial reversion of the cofactor specificity from NADH to
NADPH is observed for all the mutants (table 2). In fact, the mutants all
exhibit an NADPH-dependent activity. The apparent affinity constant
(Kma) for NADPH is of the same order for the three mutants.

[0086]The data as a whole show that the mutations made very greatly reduce
the NADH-dependent activity of the enzyme and introduce an affinity for
NADPH. The three mutants exhibit a similar affinity for NADPH. On the
other hand, the double-mutant and triple-mutant forms are particularly
advantageous because they have a better specific activity compared with
the single mutant.

[0087]In order to evaluate the impact of the change in BDH cofactor in the
enological model strain V5ald6, the BDH1 gene carrying the triple
mutation, called BDH1223, was overexpressed in this strain by in
situ site-directed mutagenesis. Two oligonucleotides, including one of 55
base pairs (SEQ ID No. 9) containing the target mutations, were
synthesized and used to amplify the loxpKanMXloxp-TDH3p-BDH1 region of a
V5ald6 BDH1 strain. The amplified fragment, containing the KanMX marker
and, under the control of the TDH3 promoter, the BDH1 gene up to the
region containing the 3 mutations, was used to transform a V5ald6 strain.

[0088]This strategy made it possible, in a single step, to integrate the
target mutations into the genomic sequence of Bdh1p, by homologous
recombination, while at the same time overexpressing the mutated gene.

[0090]The PCR product obtained was precipitated with ethanol in the
presence of salts. 4 μg of the precipitated DNA were used to transform
the S. cerevisiae V5ald6 yeast strains.

[0091]The integration was verified by enzymatic digestion of the PCR
product for 2 hours at 37° C. with the Bbs I enzyme using the
following reaction mixture: 13.5 μl of PCR product; 1.5 μl of
10×NEB2 buffer (Biolabs, USA); 0.25 μl (1.25 U) of Bbs I
(Biolabs, USA).

[0092]The enzymatic activity was then determined on the crude protein
extract of a strain having integrated the desired mutations. For each
assay, 20 μl of crude extract were used in the presence of 0.2 mM of
NADH/NADPH and 50 mM of acetoin.

[0093]In parallel, the modified region was sequenced, thereby making it
possible to verify that the mutated BDH1 sequence is identical to the
sequence of the native gene, with the exception of the mutations
introduced, which result in 3 amino acids being changed in the protein
(FIG. 3).

Physiological Impact of the Change in Cofactor Specificity of Bdh1

[0094]The V5ald6 BDH1 pGPD1 strain, overexpressing wild-type Bdh1, was
compared with the clone having the NADPH-dependent Bdh1 (V5ald6
BDH1223 pGPD1) in enological fermentation on a synthetic must at 200
g/l of glucose. These 2 strains were studied in fermentation in the
absence of uracil (selection pressure).

[0095]Under these conditions, the fermentation-rate and biomass profiles
are identical. No significant effect is observed on the fermentative
by-products, except for an increase in the formation of
α-ketoglutarate (from 480 mg/l to 620 mg/l), of OH-glutarate (from
1400 mg/l to 1620 mg/l) and of glycerol (from 30.2 g/l to 32.2 g/l) in
the strain overexpressing the mutated Bdh compared with the native Bdh.
The increase in glycerol formation can be explained by a greater
availability of NADH, owing to the preferential use of NADPH by
Bdh1223. The buildup of alpha-ketoglutarate may be linked to the
limitation of its reduction to glutamate, a reaction which is catalyzed
by NADPH-dependent glutamate dehydrogenase Gdh1p. On the other hand, the
production of OH-glutarate, another reduced product of
alpha-ketoglutarate, could be the result of the greater availability of
NADH. These observations clearly show an in vivo effect of the change in
cofactor for BDH1, from NADH to NADPH.

[0096]The most striking effect obtained with the change in cofactor
specificity is a very significant reduction in the production of acetoin
in the V5ald6 BDH1223 pGPD1 strain compared with the amount produced
by V5ald6 BDH1 pGPD1. Under the conditions tested, this reduction is
approximately 400 mg/l (FIG. 4).

[0097]The inventors furthermore showed that this effect was also obtained
in another genetic background, the S. cerevisiae laboratory strain
CEN.PK. Specifically, the overexpression of BDH223 in CEN.PK ald6
pGPD1, which produces 1638 mg/l of acetoin on a synthetic must containing
50 g/l of glucose, results in a 680 reduction in acetoin production,
whereas overexpression of the native enzyme reduces this production by
only 18%. The amount of 2,3-butanediol increases stoichiometrically as
the acetoin decreases (FIG. 5).

[0098]This clearly shows a physiological effect linked to the change in
cofactor specificity of Bdh1 at high concentrations of glycerol. Not only
is the amount of Bdh1 enzyme limiting in strains overproducing glycerol,
but also the availability of NADH.

[0099]The invention thus provides wild-type and mutated yeast strains
which overproduce 2,3-butanediol dehydrogenase, including strains which
overproduce glycerol, with controlled production of acetate, which build
up 2,3-butanediol in large amount and with a considerably reduced
production of acetoin.

[0100]As the above examples show, the overexpression of BDH1 via in situ
replacement of its promoter with that of TDH3 proves to be very
effective, since an increase in activity by a factor of 30 was obtained.
The analysis of these strains over the course of enological fermentation
shows, moreover, that the level of expression of BDH1 and of enzymatic
activity of BDH is indeed a limiting factor in the conversion of acetoin
to 2,3-butanediol in strains which overproduce glycerol.

[0101]These results show that the conversion of acetoin to 2,3-butanediol
is also limited by the availability of NADH. In fact, the overexpression
of a mutated form of BDH that is NADPH-dependent creates an additional
decrease in acetoin compared with a strain overexpressing the wild-type
enzyme.

[0102]Thus, the strategy followed according to the invention, of
overexpression of BDH1 and of change in cofactor specificity of BDH, from
NADH to NADPH, enables an extremely efficient conversion of acetoin to
2,3-butanediol.

[0103]The strategy of BDH1223 overexpression may be envisioned for
reducing the acetoin in any modified or unmodified yeast strain that
builds up this compound, such as strains which overproduce glycerol.
Another example is a strain overexpressing a bacterial NADH oxidase. It
has in fact been shown that overexpression of the NOXE gene encoding NADH
oxidase of Lactococcus lactis, in the V5 strain, decreases the
intracellular NADH content, thereby leading to a decrease in the ethanol
yield owing to the limitation of the alcohol dehydrogenase activity. In
this strain, the carbons not directed to the formation of ethanol build
up at the acetaldehyde junction, in particular acetoin (Heux et al.,
2006). The expression of a mutated form of BDH that uses NADPH as a
cofactor, promoting the conversion of acetoin to 2,3-butanediol, could
reduce the production of this compound.

[0104]The other mutants described above, which exhibit an increased
affinity with respect to NADPH, in particular the double mutant which has
the same characteristics as the triple mutant, may also prove to be
advantageous for reducing acetoin under physiological conditions.

[0105]The expression of an NADPH-dependent Bdh1p in yeast makes it
possible to modify the NADP/NADPH cofactor balance and, in this respect,
may constitute an advantageous tool in the study of the intracellular
oxidoreduction equilibrium.

BDH1 in Yeast: Interspecies and Intraspecies Sequence Homologies

[0106]The change in specificity of BDH was carried out in the S288C and V5
strains, although the cofactor binding site differs by one amino acid
between these 2 strains. The V5 strain in fact has a valine at position
222, whereas S288C has an isoleucine at the same position. In each of
these strains, the introduction of the E221S, E221S/I(V)222R and
E221S/I222R/A223S mutations proved to be effective and made it possible
to obtain a complete change in cofactor specificity from NADH to NADPH.

[0107]The conservation of this site was analyzed in other strains of
Saccharomyces cerevisiae in which the genome has been sequenced
(http://www.sanger.ac.uk/gbrowse/gbrowse/Saccharomyces/).

[0108]The study shows that the 12 strains analyzed can be divided up into
2 groups, one (DBVPG1373, DBVPG1853, DBVPG6765, L--1374,
L--1528 and SK1) characterized by the 221 EVA 223 sequence (V5-type
sequence), the other (DBVPG6044, S288C, Y55, YGPM, YPS128 and YPS606) by
the 221 EIA 223 sequence (S288C-type sequence). These observations
indicate that the effects of the site-directed mutagenesis, described
above, can extend to the other strains of S. cerevisiae.

[0109]Similarly, the sequence homologies of Bdh1p (S288C) with other yeast
species (http://cbi.labri.fr/Genolevures/path/) were searched. 5
orthologous BDH1 genes were found in Candida glabrata, Kluyveromyces
lactis and Debaryomyces hansenii. The protein sequence alignment shows a
very strong conservation of the NADH-binding site, amino acids 221 and
223 being identical to those of S. cerevisiae. The position of amino acid
222 is more variable, as observed between S. cerevisiae strains. In the
case of non-Saccharomyces yeasts, this amino acid is a proline, which is
of the same family (hydrophobic aliphatic amino acids) as isoleucine or
valine.

[0110]Owing to this sequence similarity, the amino acid changes by
site-directed mutagenesis, described above, and the effects thereof, may
be entirely applied to yeast species other than S. cerevisiae.

[0111]Other Example of Characterization of the Enzymatic Properties of the
Mutated Forms of Bdh1p

[0115]20 ml of YNB medium (2% galactose) were inoculated with the
transformants. The cells were taken in the growth phase, at OD (600 nm)
2.5. The cells (109) were suspended in 500 μl of buffer A (20 mM
sodium phosphate (pH 7.0) containing 1% glycerol and 0.5 mM DTT) and
ground using glass beads. After centrifugation at 12 000 rpm for 5 min,
the supernatant was recovered and used for the enzymatic assays.

[0116]Enzymatic Activity Assay

[0117]The 2,3-butanediol dehydrogenase activity was determined using the
crude extracts by measuring the change in absorbance at 340 nm as
described below. The assays were carried out in the presence of 33 mM of
sodium phosphate (pH 7.0), 0.5 mg/l of BSA (bovine serum albumin), of
acetoin and of 1 mM NAD(P)H. The protein concentration in the extracts
was determined with the Bradford method (Bio-Rad). The Km and
Vm for NAD(P)H (table 3) were determined in the presence of 50 mM
and 450 mM of acetoin for Bdh1 and the Bdh1 mutants, respectively.

[0119]The mutants all exhibit an NADPH-dependent activity (tables 3 and
4). The affinity of the double-mutant and triple-mutant forms for NADPH
(44 μM) is identical to the affinity of Bdh1 for NADH (45 μM). The
comparison of the specific activities of each enzyme in the presence of 1
mM NAD(P)H (table 3) shows that the triple-mutant form has a
substantially higher activity for NADPH than the double mutant.

[0123]The ENYpgi1 strain is deleted of PGI1 which encodes phosphoglucose
isomerase. This enzyme is located at the junction of glycolysis and the
pentose phosphate pathway (PPP). In this mutant, glucose-6-phosphate is
entirely redirected to the PPP, thereby generating an excess of NADPH
which prevents the strain from developing. On the other hand, this mutant
can develop if it is provided with an NADPH reoxidation system, for
example provided by expression of the E. coli transhydrogenase udha.

[0124]In fact, FIG. 6 shows that ENYpgi cannot grow on galactose as the
sole carbon source, whereas the expression of udha makes it possible to
restore its growth. If acetoin is added to the culture medium (FIG. 6c),
the growth of ENYpgi1 expressing the NADPH-dependent mutated forms of
Bdh1 is also restored. It follows from these results that the
NADPH-dependent BDH forms are capable of reoxidizing the NADPH produced
in excess by this strain, to NADP, thereby demonstrating their
functionality in vivo.

[0125]Overexpression of BDH1 in Order to Reduce Diacetyl Formation

[0126]Diacetyl (2,3-butanedione) is undesirable in fermented beverages. In
wine, its detection threshold ranges between 0.2 and 2.8 mg/l, and it is
considered to be undesirable above 5 mg/l. In beer, diacetyl poses a
major problem owing to its very low detection threshold (0.1 ppm) and a
long maturation stage is necessary in order to eliminate this compound.

[0127]Diacetyl comes from the oxidative decarboxylation of
α-acetolactate, an intermediate in the biosynthesis of isoleucine,
leucine and valine, and can be reduced to acetoin. It has been shown that
Bdh1 has, in vitro, a diacetyl-reducing activity. In order to study the
impact of Bdh1 on diacetyl in vivo, the final concentration of diacetyl
produced by the V5ald6 pGPD1 and V5ald6 BDH1 pGPD1 strains in the
experiment described in FIG. 2a was assayed.

[0128]The diacetyl concentration was determined by SPME (solid-phase
microextraction) using deuterated diacetyl-d6 as internal standard, and
GC-MS (Hayasaka & Bartowsky, 1999). The overexpression of BDH1 makes it
possible to reduce the production of diacetyl by a factor of 2 (FIG. 7).
Similar results were obtained with a strain overexpressing the
triple-mutant form of Bdh1.